The major findings of our HTLV-I studies have been: 1) Increased HTLV-I specific CD8+ cells have been shown to be elevated in the peripheral blood and CSF of HAM/TSP patients and directly proportional to the amount of HTLV-I proviral DNA and RNA. These antigen-specific T cells are considered to be immunopathogenic and may be directly involved in virus-host interactions in the CNS 2) We demonstrated that spontaneous degranulation and IFN-gamma production (defined by CD107+ IFN-gamma+ cells) was correlated with proviral DNA load in CD14+ macrophages from HTLV-I-infected patients, and that enhanced IL-15 expression on CD14+ cells mediated the dysregulation of CD8+ T cells in HAM/TSP patients. The observation of HTLV-I infected macrophages is novel and suggests a wider tropism for this virus than previously reported. The effect of HTLV-I on CD14+ macrophages is also being investigated. 3) CD4+CD25+ regulatory T cells are important in the maintenance of immunological self-tolerance and in the prevention of autoimmune diseases. We have demonstrated that in HTLV-I infected CD4+CD25+ T cells of patients with HAM/TSP the expression of the forkhead transcription factor Foxp3, a specific marker of regulatory T cells, was lower than that of healthy individuals. In addition, we are investigating the demethylated Fox3 status of HAM/TSP patients and have shown that there is decreased demethylation in PBMC and CD4+CD25+ T cells compared to healthy donors. 4) Basic laboratory investigations have demonstrated the importance of the cytokine IL-15 in the life and death of lymphocytes and for its role in autoimmune disorders such as HAM/TSP. IL-15 is pivotally involved in the survival of CD8+ memory T-cells including self-directed cells and we have shown that in HAM/TSP, IL-15 is essential for the survival of HLA class I restricted virus antigen-specific effector and memory CD8+ T-cells. These CD8+ antigen-specific CTL are thought to play a major role in the immunopathogenesis of HAM/TSP since they have been localized in brain and spinal cord sections of patients. As IL-15 is a pro-inflammatory cytokine that stimulates the production of inflammatory cytokines, the release of IL-15 induced by HTLV-I tax in patients with HAM/TSP may underlie the pathogenesis of this autoimmune disease. The mode of action of IL-15 and its receptor subunits IL-15 and IL-15Ralpha are coordinately stimulated and expressed following HTLV-I tax stimulation. We have shown that this trans-stimulation can be virtually totally inhibited for NK and CD8+ T-cells by the addition of a humanized monoclonal antibody, Hu MiKbeta1, that blocks IL-15 binding to the IL-2/IL15Rbeta; receptor subunit expressed on these cells. Collaborative research by the Viral Immunology Section, NINDS and the Metabolism Branch, NCI have initiated a phase I clinical trial for the treatment of HAM/TSP using Hu MiK-beta-1 that blocks the action of IL-15. 5) This understanding of the roles of IL-2/IL-15 in the pathogenesis of HAM/TSP has led to targeted therapeutic approaches. Additionally, given the fact that IL-2/IL-15 signal through the common gamma chain receptor, a novel potentially more effective targeted therapeutic approach to HAM/TSP might consist of blocking the common gamma chain with an antagonizing peptide. This would inhibit the action of multiple cytokines implicated in the disease and might be better tolerated than monoclonal antibodies or small molecule inhibitors. A peptide called BNZ gamma is a novel anti-cytokine peptide that was designed based on the presence of a moderately conserved region among all the cytokines sharing the common gamma chain receptor, overlapping with the D-helix. It is a 19-mer peptide (BNZ-g-001) that, based on proliferation assays in cytokine-dependent human and murine cells lines, was found to selectively block signaling of IL-2, IL-15, and IL-9. We have examined the effects of BNZ- &#947; on multiple in vitromarkers of immune activation in HAM/TSP. The peptide significantly suppressed spontaneous proliferation (SP) of the PBMCs, reduced the frequency of Tax-specific CD8+ T-cells, and the degree of STAT5 phosphorylation after 20h of culture. It was as effective as a combination of monoclonal antibodies against CD25 and CD122 in reducing SP and STAT5 phosphorylation. 6) We have developed a novel digital based PCR methodology (ddPCR) for the detection of human viruses. We have characterized PCR promer and pair probes for the detection of HTLV-I, HTLV-II, EBV, JCV, CMV and HHV6 A and HHV6B. 7) We continue to extend our work on the detection of the human herpesvirus (HHV-6) from brain resections of patients with mesial temporal lobe epilepsy and patients with neurologic complications following allogeneic bone marrow transplants. We have developed a novel electrochemiluminescent ELISA method for the quantitative detection of antibodies to HHV-6 IgG. We have screened large panels of sera from patients with MS, encephalitis and controls. Preliminary results suggest significantly more HHV-6 DNA in patients with encephalitis of unknown origin than previously reported. 8) Using a novel and highly sensitive digital droplet PCR (ddPCR) assay, we investigated the incidence of HHV-6A and HHV-6B coinfection in samples from healthy donors and MS patients. In our assessment of healthy donors, we observed a heretofore-underappreciated high frequency of coinfection in PBMC and serum, and found that our assay precisely detects both HHV-6A and HHV-6B chromosomally integrated virus. In our comparison of MS patients with healthy donors, we detected an elevated frequency of coinfection in MS saliva. Identifying and quantifying both species of HHV-6 may provide clinically relevant information, as well as enhance our understanding of the roles of HHV-6A and HHV-6B in health and disease. 9) A number of laboratory studies have demonstrated that virus infection can activate a variety of CNS-specific cells including microglia and astrocytes. The VIS has investigated the effects of both a human retrovirus (HTLV-I) and herpesvirus (HHV-6) in these cell populations, in vitro.